Abstract This application seeks continued support for a new project, based on discoveries made during a research program, funded by the NCI for over 45 years, on biology and evolution of retroviruses and their roles in AIDS and cancer, with a particular focus on Human endogenous retroviruses (HERVs). The studies described in this proposal will ccenter on a provirus in the relatively recent HERV-K (HML-2) group that integrated in a site on chromosome 3, band q12.3 of a common human/gorilla ancestor at least 8 million years ago. In prior work, we discovered that this provirus has several unique features consistent with its having evolved into a restriction factor capable of strongly blocking the virion assembly step in replication both of the reconstructed, infectious, HERV-Kcon and, remarkably, also of HIV. Adaptations to this role include an LTR mutation that leads to widespread expression in all normal human solid tissues and a mutation (or mutations) in the gag CA encoding region that lead to transdominant misassembly of the Gag precursor of the infecting virus at the cell membrane. The current proposal will follow up on these observations with the aims of obtaining a detailed understanding of the mechanism of co assembly and inhibition of HIV Gag assembly and release, understanding the evolution of the 3q12.3 provirus and the scope of its activity against other retroviruses, isolation of HIV escape mutants resistant to 3q12.3 inhibition, and testing the feasibility of using reactivation of the 3q12.3 provirus in HIV target cells as a novel method of prevention of HIV replication.